星形细胞瘤中TNKS1与Wnt/β-catenin 通路的关系及其生物学功能的研究

TNKS1 is a newly found positive regulator of the Wnt/β-catenin pathway, and compounds like XAV939 targeting TNKS1's poly-ADP-ribosylation（PARsylation）activity can inhibit growth of the APC-defective CRC cell line DLD-1 via blocks Wnt/β-catenin pathway. However, little is known about the molecular relationship between TNKS1 and Wnt/β-catenin pathway in astrocytoma. Our previous study showed that expression of TNKS1 was not only significantly increased but also correlated with pathologic grade and β-catenin activity in human astrocytoma, indicated that TNKS1 might exert its function through Wnt/β-catenin pathway in astrocytoma. In this project, the TNKS1 cDNA is cloned and transfected into human astrocytoma cell line U251, the biological effects on tumor cells, level of Phospho-β-catenin and transcriptional activity of β-catenin/TCF/LEF are analyzed. RNA interference(RNAi) is also used to determine the functions of TNKS1. Then a U251 astrocytoma model is established in nude mice to clarify the functions of TNKS1 in vivo. Our subject is designed to study the biological role of TNKS1 in astrocytoma, to clarify whether TNKS1 exert its function through Wnt/β-catenin pathway in the tumorigenesis of astrocytoma and elucidate the mechanisms involved in this process, to provide new ideas for the diagnosis and gene therapy for astrocytoma.

TNKS1是新发现的Wnt/β-catenin通路的正向调控因子，抑制其PARP活性可使Wnt通路失活并抑制结肠癌细胞的增殖。但TNKS1是否通过Wnt/β-catenin通路参与星形细胞瘤的发生发展目前尚不清楚。我们前期研究发现TNKS1在Wnt通路相关的人脑星形细胞瘤组织中异常高表达，且与其病理级别和β-catenin表达正相关，提示TNKS1在星形细胞瘤恶性进程中与Wnt/β-catenin通路相关。本课题将进一步于细胞水平研究TNKS1对β-catenin磷酸化水平和入核过程的调控及机制，阐明TNKS1通过Wnt/β-catenin通路对星形细胞瘤生物学特性的影响；并通过动物实验对体外实验结果进行验证。本研究为深入揭示星形细胞瘤的发生机制奠定基础，为星形细胞瘤的诊断及分子靶向治疗提供更有针对性的靶点和依据。

TNKS1是新发现的Wnt/β-catenin通路的正向调控因子，抑制其PARP活性可使Wnt通路失活并抑制结肠癌细胞的增殖。但TNKS1是否通过Wnt/β-catenin通路参与星形细胞瘤的发生发展目前尚不清楚。我们前期研究发现TNKS1在Wnt通路相关的人脑星形细胞瘤组织中异常高表达，且与病理级别和β-catenin表达正相关，提示TNKS1在星形细胞瘤恶性进程中与Wnt/β-catenin通路相关，具体机制仍不清楚。本课题首先构建TNKS1过表达和沉默载体并转染人脑星形胶质母细胞瘤U251细胞株，利用MTT检测各组细胞增殖情况，流式检测各组细胞凋亡和细胞周期，Transwell检测细胞侵袭能力，Western blot检测各组 TNKS1、cyclinD1、MMP-7、β-catenin、磷酸化β-catenin、c-myc表达，免疫共沉淀检测各组TNKS1 与β-catenin 的相互作用情况，结果发现与对照组相比，过表达组的U251细胞活力明显升高，细胞凋亡明显下降，G1期明显下降，S期明显升高，细胞侵袭能力明显升高，TNKS1、cyclinD1、MMP-7、β-catenin、磷酸化β-catenin、c-myc的表达都升高，而沉默组的结果相反。通过免疫共沉淀的结果发现，用β-catenin 抗体拉下后都能检测到TNKS1的表达，说明在U251细胞中TNKS1和β-catenin之间确实存在相互作用。同时建立裸鼠皮下U251细胞荷瘤模型，沉默TNKS1可明显抑制肿瘤体积增长。上述结果说明TNKS1在U251细胞中可促进β-catenin的磷酸化，抑制其降解复合物对β-catenin的降解，继之β-catenin转入细胞核内，激活Wnt/β-catenin通路下游靶基因，从而启动Wnt/β-catenin通路，因此表明TNKS1是Wnt/β-catenin通路的正向调控因子。本课题首次通过体内外实验证明了TNKSl可通过正向调控Wnt/β-catenin通路参与星形细胞瘤的恶性进程，提示了TNKS1有可能成为基因治疗人脑星形细胞瘤的新靶点，本课题为星形细胞瘤的分子靶向治疗提供了更有针对性的靶点。